A FUNGAL PROTEIN DISULFIDE ISOMERASE

Abstract: A fungal protein disulfide isomerase obtainable from fungal species belonging to Aspergillus, especially A. oryzae, or A. niger, is disclosed. Furthermore sequences for recombinant production of the protein disulfide isomerase are disclosed.

Description: Title: A Fungal Protein Disulfide Isomerase FIELD OF THE INVENTION The present invention relates to an active recombinant fungal protein disulfide isomerase, compositions comprising said fungal protein disulfide isomerase, and methods for their use; a DNA construct comprising a DNA sequence encoding said fungal protein disulfide isomerase, and a vector and cell harbouring the DNA construct. Furthermore, the present invention relates to a method of preparing the fungal protein disulfide isomerase by use of both traditional and recombinant DNA techniques. BACKGROUND OF THE INVENTION The use of protein disulfide redox agents such as protein disulfide isomerases (PDI) , and thioredoxins (TRX) for various purposes has been known for some time. Protein disulfide redox agents catalyse the general reaction: R i -SH + R 2 -SH + Enz oχ ■ " ■ R J -L-S-S-R2 + Enz red (reaction I) where R 1 and R 2 represent protein entities which are the same or different, either within the same polypeptide or in two polypeptides, Enz oχ is a protein disulfide redox agent in the oxidised state, and Enz red is a protein disulfide redox agent in the reduced state. EC 5.3.4.1 refers to an enzyme capable of capable of catalysing the rearrangement of -S-S- bonds in proteins and EC 1.6.4.4 and EC 1.8.4.2 is an example of enzymes catalysing the reaction with NAD(P)H and glutathione as a mediator, respectively. This type of activity has been designated as protein disulfide isomerase, sulfhydryl oxidase, protein disulfide reductase, disulfide isomerase, protein disulfide transhydrogenase, and sulfhydryl oxidase. Disulfide linkages in proteins are formed between cysteine residues and have the general function of stabilising the three dimensional structure of the proteins. They can be formed between cysteine residues of the same or different polypeptides. Disulfide linkages are present in many types of proteins such as enzymes, structural proteins, etc. Enzymes are catalytic proteins such as proteases, amylases, etc., while structural proteins can be scleroproteins such as keratin, etc. Protein material in hair, wool, skin, leather, hides, food, fodder, stains, and human tissue contains disulfide linkages. Treatment of some of these materials with PDI and TRX, and a redox partner have been described previously. The use of TRX for waving, straightening, removing and soften- ing of human and animal hair was described by Pigiet et al. (EP 183506 and WO 8906122) . Pigiet (US 4771036) also describes the use of TRX for prevention and reversal of cataracts. Schreiber (DE 2141763 and DE 2141764) describes the use of protein disul¬ fide transhydrogenase for changing the form of human hair. Pigiet (EP 225156) describes the use of TRX for refolding denatured proteins. Use of TRX to prevent metal catalysed oxidative damage in biological reactions is described by Pigiet et al. (EP 237189) . Toyoshima et al. (EP 277563 and EP 293793) describes the use of PDI to catalyse renaturation of proteins having reduced disulfide linkages or unnatural oxidised disulfide linkages, in particular in connection with renaturation of recombinantly produced proteins. Brockway (EP 272781) , and King and Brockway (EP 276547) describe the use of PDI for reconfiguration of human hair, and for treatment of wool, respectively. Sulfhydryl oxidase for the treatment of Ultra-high temperature sterilized milk is described in US 4894340, US 4632905, US 4081328 and US 4053644. Schreiber (DE 2141763 and DE 2141764) describes the use of protein disulfide transhydrogenase for changing the form of human hair. The uses of such enzymes have all been connected with reduction of protein disulfide linkages to free protein sulhydryl groups and/or the oxidation of protein sylfhydryl groups to protein disulfide linkages, and/or the rearrangement of disulfide linkages in the same or between different polypeptides, and sometimes to the use of these processes in sequence. Protein disulfide redox agents can be divided into two main groups of enzymes, thioredoxin type (TRX), and protein disul¬ fide isomerase type (PDI) . Both these can be modified to obtain protein engineered derivatives, chemical modifications and hybrids of TRX and/or PDI (ENG) . TRX is a 12-kDa protein having a redox-active disulfide/dithiol and catalysing thiol-disulfide exchange reactions (Edman et al., Nature 317:267-270, 1985; Holmgren, Ann. Rev. Biochem. 54:237-271, 1985; Holmgren, J. Biol. Che . 264:13963-13966, 1989) . PDI consists of two subunits, each consisting of two domains which are homologous to TRX. TRX and PDI can be obtained from a number of sources: PDI: protein disulfide iso erases have mainly been identified from mammalian sources, such as Bovine (Yamauchi et al., Biochem. Biophys. Res. Commun. 146:1485-1492, 1987), Chicken (Parkkonen et al., Biochem. J. 256:1005-1011, 1988), Human (Rapilajaniemi et al. EMBO J. 6:643-649, 1987), Mouse (Gong, et al., Nucleic Acids Res. 16:1203, 1988), Rabbit (Fliegel et al., J. Biol. Chem. 265:15496-15502, 1990), and Rat (Edman et al., Nature 317:267-270, 1985). PDI has furthermore been isolated from yeast (Tachikawa et al., J. Biochem. 110:306-313). TRX: Thioredoxin has been identified from bacteriophages, bacteria such as Escherichia coll (Wallace and Kusher, Gene 32:399-408, 1984) and Bacillus subtiliε (Chen et al . J. Biol Chem. 262:8787-8798, 1987) and eukaryotes. It would be desirable to facilitate the production of protein disulfide isomerase (PDI) , to be able of producing both larger amounts of the enzyme and to produce it in a more economical manner than what is possible by the prior art methods. Engineered variants (ENG) with improved properties for particu¬ lar applications are also highly desirable and can be prepared by a variety of methods based on standard recombinant DNA technology: 1) by using site-directed or random mutagenesis to modify the genes encoding TRX or PDI in order to obtain ENG with one or few amino acid changes, 2) by inhibiting or otherwise avoiding dimerisation of the subunits of PDI, thus giving rise to PDI monomers, 3) by producing partial monomers of PDI or TRX, in which regions of the NH2- or COOH termini of PDI or TRX are lacking, 4) by creating hybrids of PDI, TRX and/or ENG, 5) by chemically or enzymatically modifying the products of 1)- 4), 6) by a combination of any of l)-5) . ENG produced according to 1) were described by Lundstrδm et al. (J. Biol. Chem. 267:9047-9052, 1992) and by a combination of 3) and 5) by Pigiet (WO 8906122) . PDI, and TRX can, apart from their natural sources, be obtained by expression of recombinant DNA encoding plant, animal, human or microbial PDI, or TRX, in various hosts, such as microorgan¬ isms followed by purification of PDI, or TRX from extracts or supernatants of said host organisms. This goes also for ENG. Preparation of Trx from natural sources is described by Luthman and Holmgren (Biochem. 121:6628-6633, 1982), Wada and Buchanan (in "Thioredoxins, structure and function" (Gadal, Ed.) Editions du Centre National de la Recherche Scientifique) , Porque et al. (J. Biol. Chem. 245:2362-2379, 1970) and by Laurent et al . (J. Biol. Chem. 239:3436-3445), whereas recombi¬ nant production of TRX is described by Krause et al . (J. Biol. Chem. 266:9494-9500). PDI or sulfhydryl oxidase has been pre- pared from natural sources by Lambert and Freedman (Biochem J. 213:225-234, 1983), Starnes et al . (US 4632905) and Hammer et al . (US 4894340) , and by recombinant technology by among others Yamauchi et al . (Biochem. Biophys. Res. Commun. 146:1485-1492, 1987) . Finally, recombinant production of an ENG is described by Lundstrδ et al . (J. Biol. Chem. 267:9047-9052, 1992). SUMMARY OF THE INVENTION The present inventors have succeeded in cloning a DNA sequence encoding a fungal protein disulfide isomerase from filamentous fungi and in obtaining expression of an active protein disul¬ fide isomerase from said DNA sequence, both in the same species and in other organisms, especially microorganisms, and prefer- ably in fungi. Accordingly, in a first aspect the present invention relates to an active protein disulfide isomerase obtainable from filamen¬ tous fungi, specifically fungi belonging to the genus Aspergil- luε , and especially a protein disulfide isomerase obtainable from A. oryzae , or A. niger, which enzyme is immunologically reactive with an antibody raised against a purified protein disulfide isomerase derived from Aspergillus oryzae , IFO 4177, or Aspergrillus niger, A524. From the sequence of the isolated enzyme it can be seen that the protein disulfide isomerase has two -Cys-X-Y-Cys- subunits in positions 58-61 and 393-396. The invention consequently also comprises active truncated forms of the enzymes of the inven- tion, wherein at least one subunit is retained. Examples hereof could be an enzyme having an amino acid sequence corresponding to the residues 20 to 100, residues 330 to 450, or residues 360 to 430 of the appended SEQ ID No. 3, or the corresponding sequence of the enzyme of the invention in question. Under this aspect, the invention specifically relates to enzymes exhibiting protein disulfide isomerase activity comprising the amino acid residues 1-131, 1-141, 1-143, 1-163, 1-174, or 1-281, of the amino acid sequence shown in the appended SEQ ID No. 3, or variants thereof exhibiting a protein disulfide isomerase activity. Further specific enzymes are enzymes exhibiting protein disulfide isomerase activity comprising the amino acid residues 1-115, of the amino acid sequence shown in the appended SEQ ID No. 3 extended with the following sequence: Leu-Ile-Arg-Glu-Leu-Leu-Gln-Glu-Leu-Val-Asn-Lys-His-Leu; and an enzyme comprising the amino acid residues 1-511, of the amino acid sequence shown in the appended SEQ ID No. 3, and wherein the amino acid residue in position 511 is changed from Glu to Ala. In the present context, the term "derived from" is intended not only to indicate a protein disulfide isomerase produced by strains IFO 4177 or A524, but also a protein disulfide isomerase encoded by a DNA sequence isolated from these strains such as indicated in SEQ ID No. 1 and SEQ ID No. 2, or a sequence homologous thereto encoding a polypeptide with protein disulfide isomerase activity and produced in a host organism transformed with said DNA sequence. Accordingly, the present invention thus relates to an enzyme exhibiting protein disulfide isomerase activity, which enzyme is immunologically reactive with an antibody raised against a purified protein disulfide isomerase derived from Aspergillus oryzae , IFO 4177. In the present context, the term "homologue" is intended to indicate a polypeptide encoded by DNA which hybridizes to the same probe as the DNA coding for the protein disulfide isomerase enzyme under certain specified conditions (such as presoaking in 5xSSC and prehybridizing for 1 h at -40°C in a solution of 5xSSC, 5xDenhardt's solution, 50 mM sodium phos¬ phate, pH 6.8, and 50 μg of denatured sonicated calf thymus DNA, followed by hybridization in the same solution supple- mented with 50 μCi 32-P-dCTP labelled probe for 18 h at -40°C followed by washing three times in 2xSSC, 0.2% SDS at 40°C for 30 minutes) . More specifically, the term is intended to refer to a DNA sequence which is at least 70% homologous to the sequences indicated above encoding the protein disulfide isomerase of the invention. The term is intended to include modifications of the DNA sequences indicated above, such as nucleotide substitutions which do not give rise to another amino acid sequence of the protein disulfide isomerase but which correspond to the codon usage of the host organism into which the DNA construct is introduced or nucleotide substitu¬ tions which do give rise to a different amino acid sequence and therefore, possibly, a different protein structure which might give rise to a protein disulfide isomerase mutant with differ¬ ent properties than the native enzyme. Other examples of possible modifications are insertion of one or more nucleotides into the sequence, addition of one or more nucleotides at either end of the sequence, or deletion of one or more nucleo¬ tides at either end or within the sequence. In the present context the term active protein disulfide isomerase is intended to indicate an enzyme having an activity similar to that of protein disulfide isomerase, i.e. an enzyme capable of catalysing reaction I. The activity may be de¬ termined in an assay based on oxidative refolding of reduced Bowman-Birk soya bean inhibitor, e.g. as described in the Materials and Methods section below. The term "recombinant" as used about the protein disulfide isomerase of the invention is intended to indicate that it is produced by a cell transformed with a DNA sequence encoding the protein disulfide isomerase. Thus, the recombinant protein disulfide isomerase may be produced by either its parent organism or another organism. In a further aspect the present invention relates to a DNA construct comprising a DNA sequence encoding an active recom¬ binant protein disulfide isomerase of the invention as defined above. Such a DNA construct may comprise introns (an example thereof is shown in the appended SEQ ID No. 1) and/or regula¬ tory elements native to the parts coding for the mature protein disulfide isomerase of the invention, or be a cDNA construct comprising only that part coding for the mature protein disulfide isomerase (an example being the appended SEQ ID No.2) . In still further aspects the present invention relates to a re¬ combinant expression vector harbouring the DNA construct of the invention, to a cell which either harbours the DNA construct or the expression vector of the invention, and to a process for the production of a protein disulfide isomerase of the in¬ vention, wherein a cell of the invention as described above is cultured under conditions conducive to the production of the protein disulfide isomerase, and the protein disulfide isomerase is subsequently recovered from the culture. Finally, the present invention relates to compositions compris¬ ing the active protein disulfide isomerase of the invention and methods for their use in various applications. BRIEF DESCRIPTION OF THE TABLES AND DRAWING The invention is further illustrated in the accompanying tables and drawing, in which Table 1 shows an alignment of published eukaryotic PDI amino acid sequences: Bovine (Bos tauruε) (Ya auchi et al., Biochem. Biophys. Res. Commun. 146:1485-1492, 1987), chicken (Gallus gallus) (Parkkonen et al., Biochem. J. 256:1005-1011, 1988), human (Homo εapienε) (Rapilajaniemi et al. EMBO J. 6:643-649, 1987), mouse (Mus muεculuε) (Gong, et al., Nucleic Acids Res. 16:1203, 1988), rabbit (Oryctolagus cuniculuε) (Fliegel et al., J. Biol. Chem. 265:15496-15502, 1990), rat (Rattus norvegicuε) (Edman et al., Nature 317:267-270, 1985), and yeast (Saccharo- myceε cerevisiae) (Tachikawa et al., J. Biochem. 110:306-313). Table 2 shows an alignment of PDI amino acid sequences: Alfalfa (Medicago sativa) (Shorrosh and Dixon, Plant. Mol. Bio. 19:319- 321, 1992) , A. oryzae (this invention) , yeast (Saccharomyceε cereviεiae) (Tachikawa et al. , J. Biochem. 110:306-313), bovine (Boε tauruε) (Ya auchi et al., Biochem. Biophys. Res. Commun. 146:1485-1492, 1987), rat (Rattus norvegicuε) (Edman et al., Nature 317:267-270, 1985), and mouse (Mus musculus) (Gong, et al., Nucleic Acids Res. 16:1203, 1988), and Fig. 1 illustrates the construction of the expression plasmids pCaHj43l, pCaHj432, pCaHj433, and pCaHj434 further described in Example 1. DETAILED DESCRIPTION OF THE INVENTION The amino acid sequence of the protein disulfide isomerase of the invention, which was isolated from a strain of the A. oryzae , has been aligned with that of protein disulfide isomerases of other origins and have been shown to have a degree of identity of about 38% with that of Saccharomyceε cerevisiae (GenBank Ace. No. M62815) and 30% with that of Alfalfa (GenBank Ace. No. 11499). These homologies are taken to indicate that some kind of evolutionary relationship exists between protein disulfide isomerases, and that the protein disulfide isomerase of the invention may represent a distinct class of protein disulfide isomerase. It is contemplated that the protein disulfide isomerase of the invention or DNA encoding the protein disulfi¬ de isomerase may be isolated from other organisms, including animals, especially a mammal, an insect, a plant or a microor- ganisrα. In the present context, especially interesting origins are bacteria and fungi, including yeasts and filamentous fungi. As indicated above the sequence of the isolated enzyme shows 5 that the protein disulfide isomerase of the invention has two - Cys-X-Y-Cys- subunits in positions 58-61 and 393-396. The invention consequently also comprises active truncated forms of the enzymes of the invention, wherein at least one 10 subunit is retained. Examples hereof could be an enzyme having an amino acid sequence corresponding to the residues 20 to 100, residues 330 to 450, or residues 360 to 430 of the appended SEQ ID No. 3, or the corresponding sequence of the enzyme of the invention in question. 15 Under this aspect, the invention specifically relates to enzymes exhibiting protein disulfide isomerase activity comprising the amino acid residues 1-131 (SEQ ID No. 10) , 1-141 (SEQ ID No.9), 1-143 (SEQ ID No. 8), 1-163 (SEQ ID No. 7), 1- 20 174 (SEQ ID No. 6) , 1-281 (SEQ ID No. 5) , or 25-225 (SEQ ID No. 12) of the amino acid sequence shown in the appended SEQ ID No. 3, or variants/derivatives thereof exhibiting a protein disulfide isomerase activity. Further specific enzymes are enzymes exhibiting protein disulfide isomerase activity 25 comprising the amino acid residues 1-115, of the amino acid sequence shown in the appended SEQ ID No. 3 extended with the following sequence: Leu-Ile-Arg-Glu-Leu-Leu-Gln-Glu-Leu-Val-Asn-Lys-His-Leu (SEQ ID No. 11) ; and an enzyme comprising the amino acid residues 1- 30 511, of the amino acid sequence shown in the appended SEQ ID No. 3, and wherein the amino acid residue in position 511 is changed from Glu to Ala (SEQ ID No. 4) . The DNA sequence of the DNA construct of the invention encoding 35 a recombinant protein disulfide isomerase enzyme as defined above is preferably as shown in the appended SEQ ID No. 1 (genomic DNA) or SEQ ID No. 2 (cDNA) . Analogues of said sequences, which differ in one or more codons, but which encodes the recombinant protein disulfide isomerase are also within the invention. Similar DNA sequences coding for the truncated forms of the protein disulfide isomerases of the invention are also part of the invention. DNA sequences therefore can be taken from SEQ ID No. 1, or preferably SEQ ID No. 2. The DNA sequence of the DNA construct of the invention may be isolated by well-known methods. Thus, the DNA sequence may, for instance, be isolated by establishing a cDNA or genomic library from an organism expected to harbour the sequence, and screening for positive clones by conventional procedures. Examples of such procedures are hybridization to oligonucleoti- de probes synthesized on the basis of the full amino acid sequence shown in SEQ ID No. 3, or a subsequence thereof in accordance with standard techniques (cf. Sambrook et al., 1989) , and/or selection for clones expressing a protein disulfide isomerase activity as defined above, and/or selection for clones producing a protein which is reactive with an antibody raised against the protein disulfide isomerase comprising the amino acid sequence shown in SEQ ID No. 3 and in particular amino acid residues 1-143 thereof as shown in SEQ ID No. 8. A preferred method of isolating a DNA construct of the inven¬ tion from a cDNA or genomic library is by use of polymerase chain reaction (PCR) using degenerate oligonucleotide probes prepared on the basis of the amino acid sequence of the protein disulfide isomerase of the invention comprising amino acid residues 1-515 of SEQ ID No. 3. For instance, the PCR may be carried out using the techniques described in US Patent No. 4,683,202 or by R.K. Saiki et al. (1988). Alternatively, the DNA sequence of the DNA construct of the invention may be prepared synthetically by established standard methods, e.g. the phosphoa idite method described by Beaucage and Caruthers (1981) , or the method described by Matthes et al. (1984) . According to the phosphoa idite method, oligonucleoti- des are synthesized, e.g. in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors. Finally, the DNA construct may be of mixed genomic and synthe¬ tic, mixed synthetic and cDNA or mixed genomic and cDNA origin prepared by ligating fragments of synthetic, genomic or cDNA origin (as appropriate) , the fragments corresponding to various parts of the entire recombinant DNA molecule, in accordance with standard techniques. DNA constructs coding for the truncated forms of the enzyme of the invention may naturally be made in corresponding ways. The recombinant expression vector carrying the DNA construct of the invention may be any vector which may conveniently be subjected to recombinant DNA procedures, and the choice of vector will often depend on the host cell into which it is to be introduced. Thus, the vector may be an autonomously repli- eating vector, i.e. a vector which exists as an extrachromo- somal entity, the replication of which is independent of chromosomal replication, e.g. a plasmid, a bacteriophage or an extrachro osomal element, minichromosome or an artificial chromosome. Alternatively, the vector may be one which, when introduced into a host cell, is integrated into the host cell genome and replicated together with the chromosome(s) into which it has been integrated. In the vector, the DNA sequence should be operably connected to a suitable promoter sequence. The promoter may be any DNA sequence which shows transcriptional activity in the host cell of choice and may be derived from genes encoding proteins either homologous or heterologous to the host cell. Examples of suitable promoters for directing the transcription of the DNA construct of the invention, especially in a bacterial host, are the promoter of the lac operon of E. coli , the Streptomyceε coelicolor agarase gene dagrA promoters, the promoters of the Bacilluε licheniformiε α-amylase gene (amyl>) , the promoters of the Bacilluε εtearothermophiluε maltogenic amylase gene (amyK) , the promoters of the Bacilluε Amyloliguefacienε α-amylase (amyQ) , the promoters of the Bacilluε εubtiliε xylA and xylB genes etc. For transcription in a fungal host, examples of useful promoters are those derived from the gene encoding A. oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, A. niger neutral α-amylase, A. niger acid stable α-amylase, A. niger glucoamylase, Rhizomucor miehei lipase, A. oryzae alkaline protease, A . oryzae triose phosphate isomerase or A. nidulanε acetamidase. The expression vector of the invention may also comprise a suitable transcription terminator and, in eukaryotes, poly- adenylation sequences operably connected to the DNA sequence encoding the recombinant protein disulfide isomerase of the invention. Termination and polyadenylation sequences may suitably be derived from the same sources as the promoter. The vector may further comprise a DNA sequence enabling the vector to replicate in the host cell in question. Examples of such sequences are the origins of replication of plasmids pUC19, pACYC177, pUBHO, pE194, pAMBl and pIJ702. The vector may also comprise a selectable marker, e.g. a gene the product of which complements a defect in the host cell, such as the dal genes from B . subtilis or B .licheniformis, or one which confers antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracyclin resistance. Examples of Aspergillus selection markers include amdS, argB, niaD and sC, a marker giving rise to hygromycin resistance. Furthermore, the selection may be accomplished by co-transformation, e.g. as described in WO 91/17243. While intracellular expression may be advantageous in some respects, e.g. when using certain bacteria as host cells, it is generally preferred that the expression is extracellular. The protein disulfide isomerase of the invention or truncated forms thereof comprising the amino acid sequences shown in the SEQ ID Nos. 3 to 12 may furthermore comprise a preregion permitting secretion of the expressed protein disulfide isomerase into the culture medium. If desirable, this preregion may be native to the protein disulfide isomerase of the invention or substituted with a different preregion or signal sequence, conveniently accomplished by substitution of the DNA sequences encoding the respective preregions. The procedures used to ligate the DNA construct of the inven- tion, the promoter, terminator and other elements, respect¬ ively, and to insert them into suitable vectors containing the information necessary for replication, are well known to persons skilled in the art (cf., for instance, Sambrook et al. (1989) ) . The cell of the invention either comprising a DNA construct or an expression vector of the invention as defined above is advantageously used as a host cell in the recombinant produc¬ tion of a polypeptide of the invention. The cell may be transformed with the DNA construct of the invention, conveni¬ ently by integrating the DNA construct in the host chromosome. This integration is generally considered to be an advantage as the DNA sequence is more likely to be stably maintained in the cell. Integration of the DNA constructs into the host chromo- some may be performed according to conventional methods, e.g. by homologous or heterologous recombination. Alternatively, the cell may be transformed with an expression vector as described above in connection with the different types of host cells. The cell of the invention may be a cell of a higher organism such as a mammal, an avian, an insect, or a plant cell, but is preferably a microbial cell, e.g. a bacterial or a fungal (in¬ cluding yeast) cell. Examples of suitable bacteria are gram positive bacteria such as Bacillus subtilis , Bacillus licheniformiε , Bacillus lentus, Bacilluε brevis , Bacillus εtearothermophiluε, Bacilluε alkalo- philuε , Bacilluε amyloliquefaciens , Bacillus coagulanε , Bacillus circulanε , Bacilluε lautuε , Bacilluε megaterium , Ba¬ cillus thuringienεis, or Streptomyceε lividanε or Streptomyceε murinuε, or gram negative bacteria such as E . coli . The trans¬ formation of the bacteria may for instance be effected by protoplast transformation or by using competent cells in a manner known per se . The yeast organism may favourably be selected from a species of Saccharomyces or Schizosaccharomyces, e.g. Saccharomyceε cereviεiae . The filamentous fungus may advantageously belong to a species of Aεpergilluε, e.g. Aspergilluε oryzae or Aεpergil- luε niger. Alternatively, a strain of a Fuεariu species, e.g. F . oxysporum , can be used as a host cell. Fungal cells may be transformed by a process involving protoplast formation and transformation of the protoplasts followed by regeneration of the cell wall in a manner known per se . A suitable procedure for transformation of Aspergillus host cells is described in EP 238 023. A suitable method of transforming Fusarium species is described by Malardier et al., 1989. In a yet further aspect, the present invention relates to a method of producing a recombinant protein disulfide isomerase of the invention, which method comprises cultivating a host cell as described above under conditions conducive to the production of the protein disulfide isomerase and recovering the protein disulfide isomerase from the cells and/or culture medium. The medium used to cultivate the cells may be any conventional medium suitable for growing the host cell in question and obtaining expression of the protein disulfide isomerase of the invention. Suitable media are available from commercial suppliers or may be prepared according to published recipes (e.g. in catalogues of the American Type Culture Collection) . The resulting protein disulfide isomerase may be recovered from the medium by conventional procedures including separating the cells from the medium by centrifugation or filtration, if necessary after disruption of the cells, precipitating the proteinaceous components of the supernatant or filtrate by means of a salt, e.g. ammonium sulphate, followed by purifi¬ cation by a variety of chromatographic procedures, e.g. ion exchange chromatography, affinity chromatography, or the like. It is of course also possible to produce the protein disulfide isomerases of the invention by culturing the filamentous fungal natural host or parent organism of interest and recovering the protein disulfide isomerase from the culture broth in tradi¬ tional ways. The present invention also relates to compositions comprising the protein disulfide isomerase of the invention. The compositions may suitably contain 0.01-200 mg of enzyme protein per gram, preferably 0.01-20 mg of enzyme protein per gram, especially 0.01-2 mg of enzyme protein per gram, or alternatively 0.02-0.2 mg of enzyme protein per gram, or 0.01- 0.2 mg of enzyme protein per gram. The compositions of the invention may contain other ingredients known in the art as e.g. excipients, stabilizers, fillers, detergents, etc. The compositions of the invention may be formulated in any convenient form, e.g. as a powder, paste, liquid or in granular form. The enzyme may be stabilized in a liquid by inclusion of enzyme stabilizers. Usually, the pH of a solution of the composition of the invention will be 5-10 and in some instances 7.0-8.5. Other enzymes such as proteases, cellulases, oxidases, peroxidases, amylases or Upases may be included in the compositions of the invention, either separately or in a com¬ bined additive. The compositions of the invention can be used for the treatment or degradation of scleroproteins, especially hair, skin and wool, dehairing and softening of hides, treatment and cleaning of fabrics, as additives to detergents, thickening and gelation of food and fodder, strengthening of gluten in bakery or pastry products, and as pharmaceuticals for the alleviation of eye sufferings. The present invention is further illustrated in the following examples which should not, in any manner, be considered to limit the scope of the present invention. MATERIALS AND METHODS Strains Aεpergilluε oryzae IFO 4177 available from Institute for Fermentation, Osaka; 17-25 Juso Hammachi 2-Chome Yodogawa-Ku, Osaka, Japan. Aspergillus niger A524 (ATCC 16882) Aspergillus niger TSA 1. E. coli DH5αF' Determination of PDI activity The PDI is assayed using the insulin reduction assay described by James et al . , Cell 67:581-589, 1991. Plasmids pUC 19, pMT 1560, pToC 90 EXAMPLES EXAMPLE 1 Cloning of Aspergillus oryzae and Aspergillus niger PDI encoding genes 1.1. Design of oligo nucleotides for PCR amplification PDI from different organisms are highly homologous especially near the active site residues. In Fig. 1, the following 7 PDI gene products are aligned: Bovine (Bos tauruε) PDI (Ya auchi et al., Biochem. Biophys. Res. Commun. 146:1485-1492, 1987), Chicken (Gallus gallus) PDI (Parkkonen et al., Biochem. J. 256:1005-1011, 1988), Human (Homo sapiens) PDI (Rapilajaniemi et al. EMBO J. 6:643-649, 1987), Mouse (Mus muεculus) PDI (Gong, et al. , Nucleic Acids Res. 16:1203, 1988), Rabbit (Oryctolaguε cuniculuε) PDI (Fliegel et al., J. Biol. Chem. 265:15496-15502, 1990), Rat (Rattus norvegicuε) PDI (Edman et al.. Nature 317:267-270, 1985), Yeast (Saccharomyceε cerevisiae) PDI (Tachikawa et al., J. Biochem. 110:306-313). Each subunit contains two active centres (Freedman et al.. Cell 57:1069-1072, 1989) and the homology in the surroundings of these active centres are particularly strong. A consensus amino acid sequence for the active centre closest to the N-terminus was determined from the alignment as -APWCGHCK-, and an oligo deoxyribonucleotide encoding the peptide -WCGHCK- and extended with an EcoRI site in the 5 - end, was synthesized: 5 TGGAATTCTGGTGYGGNCAYTGYAA3* (primer 4762, 25 nucleotides, 32 species, SEQ ID No. 13) (Y=C or T; R=A or G; N=A, T, C, or G) . A consensus amino acid sequence for the active centre closest to the C-terminus was determined: -YAPWCGHCK-, and an oligo deoxyribonucleotide encoding the peptide -YAPWCG- in antisense and extended with a BamHI site in the 5 λ end was synthesized: 5 Λ TGGGATCCRCACCANGGNGCRTA3 (primer 4763, 23 nucleotides, 64 species, SEQ ID NO. 14) . These oligo deoxyribonucleotides (primers 4762 and 4763) were used as primers in a PCR reaction to amplify PDI-encoding gene fragments from genomic DNA from A. oryzae and A. niger. 1.2 Amplification and cloning of fragments of PDI-encoding genes. Genomic DNA was prepared from Aεpergilluε oryzae IFO 4177 and 5 Aεpergilluε niger A524 as described by Yelton et al. (Proc. Natl. Acad. Sci. USA 81:1470-1474, 1984). PCR reaction mixtures contained Taq DNA polymerase buffer supplied by Clontech laboratories Inc. and diluted as 10 described, 250 μM of each of dATP, dCTP, dGTP, and, dTTP, 100 pmol of each of primers 4762 and 4763, and 0.5 μg of genomic DNA of either A. niger or A. oryzae . The total reaction volume was 0.1 ml, and it was covered with 0.05 ml paraffin oil. 15 The following program was run on a Cetus Perkin Elmer thermal cycler: 1. cycle: 94°C for 2 min., (when the temperature reached 94°C 2.5 U of Taq DNA polymerase supplied by Clontech laboratories Inc. was added) . 20 10 cycles: 94°C for 1 min., 50°C for 1 min., and 72°C for 2 min. 30 cycles: 94°C for 1 min., 55°C for 1 min., and 72°C for 2 min. 1 cycle: 72°C for 5 min. 25 The reaction mixtures were loaded on an agarose gel, and both the A. oryzae and the A . niger DNA produced fragments of approximately 1.1 kb. 30 The fragments were digested with EcoRI and BamHI and ligated to pUC19 (Yanisch-Perron et al., Gene 33:103-119, 1985). The ligation mixture was transformed into E. coli DH5αF (Woodcock et al., Nucleic Acids Res. (1989) 17:3469-3478). Recombinant plasmids were subjected to sequence analysis using the Se- 35 quenase™ kit (United States Biochemical) and a M13 universal primer following the manufacturers instructions. The analysis confirmed that both in the case of A. oryzae and in that of A. niger sequences homologous to other PDI genes were amplified and cloned. 1.3 Genome cloning of the A . oryzae PDI-encoding gene. Genomic DNA from A. oryzae was digested with the following restriction enzymes supplied by New England Biolabs Inc. : Hindlll, BamHI, BamHI+Hindlll, EcoRI, EcoRI+HindIII, Sail, Sall+Hindlll, Bglll, Bglll+Hindlll, PstI and Pstl+Hindlll. After digestion, the reaction mixtures were run on a 1% agarose gel and then blotted onto an Immobilon N™ membrane (Millipore Corporation) following the manufacturers instructions. The membrane was probed with the cloned A . oryzae PCR product isolated as a BamHI-EcoRI fragment and radio labelled with 32 P. After stringent washes the membrane was subjected to autora- diography. Genomic DNA from A. niger was digested with the following restriction enzymes: Bglll, BamHI, BamHI+Bglll, EcoRI, EcoRI+BglII, Sail, Sall+Bglll, Hindlll, Hindlll+Bglll, PstI and Pstl+Bglll. The Southern blot was made as described with A. oryzae , only the A. niger PCR product was used as probe. 1.4 Construction of genomic A . oryzae library. Southern analysis indicated that the A . oryzae PDI gene was located on a 6.8 kb Bglll fragment. Genomic A. oryzae DNA was digested with Bglll and fragments ranging from 5 kb to 8.5 kb were isolated from an agarose gel. Subcloning thereof and Southern analysis indicated that the A. oryzae PDI gene was located on a 2.3 kb BamH I, Hind III fragment. Genomic A. oryzae DNA was digested with BamH I and Hind III and fragments ranging from 1.9 - 3 kb were isolated from an agarose gel. This mixture of fragments was ligated to pUC19 digested with BamHI and Hind III. The ligation mixture was used to transform E. coli DH5αF Λ . The transformed E . coli cells were spread onto 10 agar plates using ampicillin selection. 1.5 Screening of the A. oryzae genomic library. The libraries were screened using the filter colony hybridiza¬ tion method described by Gergen et al. (Nucleic Acids Res. 7:2115-2136, 1979). The probe that was used for the Southern blot was also used for the colony hybridization. Positive clones were isolated and confirmed by sequence analysis using sequencing primers designed from the sequences of the PDI fragments. One of the plasmids containing the desired fragment was termed pCaHj 425. 1.6 Sequence of the gene. The gene was sequenced using the Tag DyeDeoxy™ Terminator cycle sequencing kit supplied by Applied Biosystems following the manufacturers instructions. The sequence reactions were run on an Applied Biosystems 373A DNA sequencer and the data were evaluated using the Macintosh computer program SegEd version 1.0 supplied by Applied Biosystems. The sequence of the A. oryzae gene is shown in the appended SEQ ID No 1. The amino acid composition of the purified PDI obtained as described in Example 2 was in accordance with the composition deduced from the DNA-sequence shown in SEQ ID No. l. From homology to other PDI genes and consensus splicing sequences a cDNA sequence as shown in SEQ ID no. 2 was suggested. The derived protein sequence is as shown in SEQ ID No. 3. EXAMPLE 2. Expression of truncated forms of the A. oryzae PDI gene. 2.1 Construction of expression plasmids. The PDI gene of A. oryzae was truncated at various positions by introduction stop codons. This was done by PCR amplification of the PDI gene using a 5' PCR primer harbouring a BamH I site at its 5' end and 8 different 3' primers corresponding to 8 different truncations each harbouring a Hind III site. The sequence of the 5' primer was: 5' TTCGGATCCACCATGCGGACTTTCGCACC 3' 5205. (SEQ ID No. 15) The sequences of the eight 3' primers were: 5 ' CCAAGCTTTAGAGATGCTTGTTGACAAGCTCCTG GAGGAGCTCCCTGATAAGCTT 3' 5215. (SEQ ID No. 16) 5' CCAAGCTTTAGACCATGTATGACACAATCGCCTCG GTCTGACGAG 3' 5397. (SEQ ID No. 17) 5' CCAAGCTTTAGACAGGGGACACAGCAGGTAG 3' 5895. (SEQ ID No. 18) 5' CCAAGCTTTATGGGGTGACAGGGGACA 3' 5399. (SEQ ID No. 19) 5' CCAAGCTTTAAGACGCGATATAACCAATAAC 3' 5894. (SEQ ID No. 20) 5' CCAAGCTTTAAGTGGTGAATATATCATTGGC 3' 5893. (SEQ ID No. 21) 5' CCAAGCTTAGTGTTTCTCGGCGATGAACTT 3' 6314. (SEQ ID No. 22) 5' CCAAGCTTTACGCAGACTTGTCATCGCTAGT 3' 5204. (SEQ ID No. 23) Primer 5215 directed an extension of the PDI gene amino acid 1- 115 with the sequence Leu-Ile-Arg-Glu-Leu-Leu-Gln-Glu-Leu-Val- Asn-Lys-His-Leu; followed by a stop codon. Primer 5397 introduced a stop codon after amino acid 131. Primer 5895 introduced a stop codon after amino acid 141. Primer 5399 introduced a stop codon after amino acid 143. Primer 5894 introduced a stop codon after amino acid 163. Primer 5893 introduced a stop codon after amino acid 174. 5 Primer 6314 introduced a stop codon after amino acid 281. Primer 5204 introduced the mutation E511A (meaning substituting and a stop codon after amino acid 511. The expression plasmids were constructed by PCR amplification 10 using primer 5205 in combination with either 5215, 5397, 5895, 5399, 5894, 5893, 6314 or 5204 and pCaHj 425 as template using standard PCR conditions. The generated PCR fragments were digested with BamH I and Hind III and inserted into pMT 1560 (described in e.g. PCT/DK94/00138) digested with the same 15 enzymes (See figure 3) . The constructed plasmids were named pCaHj 432 (from primer 5215) , pCaHj 433 (from primer 5397) , PCaHj 441 (from primer 5895) , pCaHj 434 (from primer 5399) , pCaHj 440 (from primer 5894), pCaHj 439 (from primer 5893), pCaHj 445 (from primer 6314) and pCaHj 431 (from primer 5204) . 20 2.2 Transformation of A . oryzae IFO 4177. Each of the plasmids pCaHj 432, pCaHj 433, PCaHj 441, pCaHj 25 434, pCaHj 440, pCaHj 439, pCaHj 445 and pCaHj 431 were transformed into A. oryzae IFO 4177 by cotransfor ation with the amdS selection plasmid pToC 90 (described in W091/17243) following the procedure described in the published EP patent application No. 238 023. 30 A number of transformants of each plasmid were evaluated. 2.3 Transformation of A . niger TSA 1. 35 Each of the plasmids PCaHj 441, pCaHj 434, pCaHj 440 and pCaHj 439 were transformed into A. niger TSA 1 by the same procedure as with A oryzae. A number of transformants of each plasmid were evaluated. EXAMPLE 3 Fermentation purification and characterization of the Aspergil¬ lus oryzae PDI truncations. 3.1 A . oryzae IFO 4177 transformants. Crude truncated PDI preparation was isolated from supernatants obtained by fermentation of the A. oryzae or A. niger pCaHj 432, pCaHj 433, PCaHj 441, pCaHj 434, pCaHj 440, pCaHj 439, pCaHj 445 or pCaHj 431 transformants in shake flasks contain- ing YPM medium (1 liter: 5 g Difco Yeast extract, 10 g Difco peptone, 20 g maltose) . The supernatant was recovered by filtration. The PDI truncation gene products were partially purified using a 1 ml HiTrap Q™ anion exchanger from Pharmacia LKB Biotechnology AB Uppsala, Sweden following the manufac- turers instructions. Fractions were collected and analyzed by measuring the disulphide isomerase activity and by SDS PAGE. The pCaHj 434 transformants secreted a protein of approx 14 kD (SDS PAGE) not present in supernatants of the untransformed strain. Enrichment of this protein by ion exchange was followed by increased disulphide isomerase activity. The approx. 14 kD band was blotted from an SDS Page gel and subjected to N- terminal amino acid sequence determination using an Applied Biosystems 473A protein sequencer. A sequence of 7 amino acids could unambiguously be determined as: Thr-Ala-Glu-Ala-Pro-Ser- Asp. This sequence corresponds to residue 24-30 of the A. oryzae protein sequence. The size of the truncation expected from the amino acid sequence is thus 13.2 kD. So it can be concluded that the pCaHj 434 gene product is secreted to the supernatant, that it has the expected size and that it is catalytic active. The pCaHj 441 transformants secreted a protein of the same size as the pCaHj 434 transformants. Also for this truncation enrichment of the protein was followed by increased disulphide isomerase activity demonstrating that the pCaHj 441 gene product is a catalytic active secreted protein. The pCaHj 440 transformants secreted a protein of approx 16 kD not present in the untransformed strain. The expected size is 15.7 kD assuming the same N-terminal sequence as the pCaHj 434 product. Enrichment of the protein by ion exchange was followed by increased disulphide isomerase activity demonstrating that also the pCaHj 440 gene product is a catalytic active secreted protein. The pCaHj 445 transformants secreted a protein of approx 30 kD not present in the untransformed strain. The expected size is 28.6 kD assuming the same N-terminal sequence as the pCaHj 434 product. Enrichment of the protein by ion exchange was followed by increased disulphide isomerase activity demonstrating that the pCaHj 440 gene product is a catalytic active secreted protein. 3.2 A. niger TSA 1 transformants. Transformants of PCaHj 441, pCaHj 434, pCaHj 440 and pCaHj 439 were evaluated in the same way as the corresponding A. oryzae transformants with the exception that the N-terminal amino acid sequence was not determined for any of the proteins secreted by A. niger. In all other aspects the same results were obtained with the A. niger transformants as with the A. oryzae transformants. However the fermentation yield of the truncations were gen- erally lower in A . niger than in A . oryzae . Table 1 50 Pdi_MOUS8 MLS RALLCLALAW AARVGADALΞ EEDNVLVLKX SNFΞΞAIiAAH Pdi_Rat MLS RALLCLALAW AARVGADALΞ EEDNVLVLKK SNFAΞALAAK Pdi_3θvir. MLR RALLCLALTA LFRAGAGAPD ΞEDHVLVLKK GNFDEALAAH Pdi_Humaπ MLR RALLCLAVAA LVR..ADAPE EΞDHVLVLRK SNFAΞALAAH Pdi_Ra ic MLR RAVLCLALAV TA.GWAWAAΞ ΞEDNVLVLKS SNFAΞΞLAAK Pdi_Cϊlick EPLΞ EEDGVLVLRA ANFΞQALAAH Pdi_Yeast MKFSAGAVLS WSSLLLASSV FAQQEAVAPΞ DSA.WKLAT DSFNΞYIQSK 51 100 Pdi_Mous e KYLLVΞFYAP WCGHCKALAP EYAXHAAKLK AEGSΞIRLAX VDATΞESDLA Pdi_Rat NYLLVΞFYA? WCGHCKALAP EYAXAAAKLK AEGSEIRLAX VDATΞΞSDLA Pdi_Bovin KYLLVΞFYAP WCGHCKALAP EYAKAAGKLK AEGSΞIRLAK VDATΞΞSDLA Pdi_Hu_nar. KYLLVEFYAP WCGHCKALAP ΞYAKAAGKLK AEGSEIRLAX VDATEΞSDLA Pdi_Rai.it KHLLVΞFYAP WCGHCKALAP EYAKAAGKLK AEGSDIRLAK VDATΞΞSDLA Pdi_Chick RHLLVEFYAP WCGHCKALAP EYAKAAAQLK AEGSΞIRLAK VDATEEASLA Pdi Yeast DLVLAΞFFAP WCGHCKNMA? ΞYVXAAΞTL. -VEKNITLAQ IDCTENQDLC 101 150 Pdi_ _Mouse QQYGVRGYPT IKFFKNGDTA SPKΞYTAGRE ADDIVNWLKX RTGPAATTLS Pdi " _Rat QQYGVRGYPT IKFFKNGDTA SPKΞYTAGRE ADDIVNWLKK RTGPAATTLS Pdi] _Bovin QQYGVRGYPT IKFFKNGDTA SPKΞYTAGRE ADDIVNWLKK RTGPAASTLS Pdi] _Human QQYGVRGYPT IKFFRNGDTA SPKΞYTAGRE ADDIVNWLKK RTGPAATTLR Pdi] _Rabit QQYGVRGYPT IKFFKNGDTA SPKΞYTAGRΞ ADDIVNWLKX RTGPAATTLA Pdi_ _Chick QQFGVRGYPT IKFFRNGDKA APRΞYTAGRΞ ADDIVSWLKK RTGPAATTLT Pdi " Yeast MΞHNIPGFPS LKIFKNSDVN NSIDYEGPRT AΞAIVQFMIK QSQPAVAWA 151 200 Pdi_Mouse DTAAAESLVD SSΞVTVTGFF KDVΞSDSAKQ FLIxAAΞAIDD IPFGITSNSG Pdi_Rat DTAAAΞSLVD SΞSVTVIGFF -S__J.n.GSSS Q FLLLAAΞAVDD IPFGITSNSD Pdi_Bovin DGAAAΞALVΞ SSΞVAVIGFF KDMESDSAKQ FFLAAΞVIDD IPFGITSNSD Pdi_Human DGAAAΞSLVΞ SSΞVAVIGFF KDVΞSDSAKQ FLQAAΞAIDD IPFGITSNSD Pdi_Rat.it DSAAAΞSLVΞ SSΞVAVIGFF KDVΞSDAAKQ FLLAAΞATDD IPFGLTASSD Pdi_Chick DAAAAΞTLVD SSEVWIGFF KDVTSDAAKE FLLAAΞSVDD IPFGISSSAD Pdi Yeas" DLPAYLANET FVTPVIVQSG KIDADFNATF YSMANKHFND YDFVSAΞNAD 201 250 Pdi_Mouse VFSKYQLDKD GWLFKKFDΞ GR..NNFEGΞ ITKΞKLLD.F IKHNQLPLVI Pdi_Rat VFSKYQLDKD GWLFKKFDΞ GR..NNFΞGE ITKΞKLLD.F IKHNQLPLVI Pdi_Bovin VFSKYQLDKD GWLFKXFDΞ GR..NNFEGΞ VTKΞKLLD. IKHNQLPLVI Pdi_Hu_τ_an VFSKYQLDKD GWLFKKFDΞ GR..NNFEGΞ VTKENLLD.F IKHNQLPLVI Pdi_Rabit VFSRYQVHQD GWLFKXFDE GR..NNFΞGΞ VTKEKLLD.F IKHNQLPLVI Pdi_Chick VFSKYQLSQD GWLFKKFDΞ GR..NNFΞGD LTKDNLLN.F IKSNQLPLVI Pdi Yeast ..DDFKL... SIYLPSAMDΞ P YNGKKAD IADAD FΞKW LQVΞALPYFG 251 300 Pdi_Mouse ΞFTEQTAPKI FGGΞIKTHIL LFLPKSVSDY DGKLSSFKRA AΞGF..KGKI Pdi_Rat ΞFTΞQTAPKI FGGΞIKTHIL LFLPKSVSDY DGKLSNFKKA AΞGF..KGKI Pdi_Bovin ΞFTΞQTAPKI FGGΞIKTHIL LFLPKSVSDY ΞGKLSNFKKA AΞSF..KGKI Pdi_Human ΞFTΞQTAPKI FGGΞIKTHIL LFLPKSVSDY DGKLSNFKTA AΞS ..KGKI Pdi Ra it ΞFTEQTAPKI FGGFIKTHIL LFLPRSAADH DGKLSGFKQA AΞGF..KGKI Pdi_Chick ΞFTΞQTAPKI FGGΞIKTHIL LFLPKSVSDY ΞGKLDNFKTA AGN ..KGKI Pdi Yeast ΞIDGSVFAQY VΞSGLPLGYL FY ND EΞELΞΞYKPL FTELAXKNRG table 1 (continued) 301 350 Pdi_Mouse LFIFIDSDKT DN RILEFFG LKKΞECPAVR LITLΞΞEM TKY ?di_Rac LFIFIDSDKT DNQRILEFFG LKKΞECPAVR LITLΞEEM TKY Pdi_3ovir. LFIFIDSDKT DNQRILEFFG LKKΞΞCPAVR LITLΞEEM TKY Pdi_Humar_ LFIFIDSDHT DNQRILEFFG LKKΞΞCPAVR LITLEEEM TKY Pdi_Rώi LFIFIDSDHA DNQRILEFFG LKKΞECPAVR LITLEEEM TKY Pdi_Chick LFIFIDSDHS DNQRILEFFG LKKΞΞCPAVR LITLEEEM TKY Pdi Ysasc LMNFVSIDAR KFGRHAGNLN M.KΞQFPLFA IKDMTEDLKY GLPQLSΞ- 351 400 Pdi_Mouse KPESDΞLTAΞ K..ITΞFCHR FLΞGKIKPHL MΞQΞVPΞD D KQPVKVLVGA Pdi_Rac KPΞSDΞLTAΞ K..ITQFCKK FLEGKIKPKL MSQELPΞDWD KQPVKVLVGK Pdi_Bovi_ι KPESDΞLTAE K..ITΞFCKR FLΞGKIKPHL MSQΞLPDDWD KQPVKVLVGK Pdi_Huπ.ar. KPΞSΞΞLTAΞ R..ITEFCKR FLΞGKIKPHL MSQΞRAGDWD KQPVKVPVGK Pdi Ra-bi KPΞSDΞLTAΞ G..ITΞFCQR FLΞGKIKPHL MSQΞLPΞD D RQPVKVLVGK Pdi_Chic:< KPΞSDDLTAD K..IKΞFCNK FLΞGKIKPKL MSQDLPEDWD KQPVKVLVGK Pdi Yeas- DΞLSDKIVLE SKAIΞSLVKD FLKGDASPIV KSQEIFENQD S.SVFQLVGK 401 450 Pdi_Mouse NFΞΞVAFDΞK KNVFVEFYA? WCGKCKQLAP IWDKLGETY. KDHENIIIAK Pdi_Rat NFΞΞVAFDEK KNVFVEFYA? WCGHCKQLAP IWDKLGETY. KDHENIVIAK Pdi_Bovir_ NFEEVAFDΞK KNVFVΞFYAP WCGHCKQLAP IWDKLGETY. KDHΞNIVIAK Pdi_Human NFΞDVAFDΞK KNVFVEFYA? WCGHCKQLAP IWDKLGETY. KDHENIVIAK Pdi_Rabic NFΞΞVAFDEK KNΓVFVΞFYAP WCGHCKQLAP IWDKLGETY. KΞKQDIVIAK Pdi_Chick NFΞΞVAFDΞN KNVFVΞFYAP WCGHCKQLAP IWDKLGETY. RDHΞNIVIAK Pdi Yeas- NHDEIVNDPK KDVLVLYYA? WCGHCKRLAP TYQELADTYA NATSDVLIAK 451 500 Pdi_Mous e MDSTANΞVΞA VKVHΞFPTLK FFPASADRTV IDYNGERTLD GFKKFLΞSGG Pdi_Rat_ MDSTANΞVΞA VKVHSFPTLK FFPASADRTV IDYNGΞRTLD GFKKFLESGG Pdi_Bovin MDSTANEVΞA VKVHSFPTLK FFPASADRTV IDYNGERTLD GFKKFLΞSGG Pdi_Human MDSTANEVΞA VKVHSFPTLK FFPASADRTV IDYNGERTLD GFKKFLΞSGG Pdi_Raiιit_ MDSTANΞVΞA VKVHSFPTLK FFPAGPGRTV IDYNGERTLD GFKKFLΞSGG Pdi_Chick MDSTANΞVEA VKIHSFPTLK FFPAGSGRNV IDYNGΞRTLΞ GFKKFLΞSGG Pdi Yeas - LDHTENDVRG WIΞGYPTIV LYPGGKKSES WYQGSRSLD SLFDFIKΞNG 501 538 Pdi_Mouse QDGAGDDEDL •DLEΞ..ALE PDMΞΞ..DDD QKAVKDEL Pdi_Rat QDGAGDNDDL •DLEΞ..ALΞ PDMΞΞ..DDD QKAVKDΞL Pdi_3ovi_ι QDGAGDDDDL ΞDLΞΞ..AΞΞ PDLΞΞ..DDD QKAVKDEL Pdi_Human QDGAGDDDDL ΞDLΞΞ..AΞΞ PDMΞΞ..DDD QKAVKDEL Pdi_Ra ic QDGAGDΞDGL EDLΞΞ..AΞΞ PDLΞΞ..DDD QKAVRDΞL Pdi_Chick QDGAAADDDL ΞDLΞT..DEE TDLΞΞGDDDΞ QKIQKDEL Pdi Yeasc HFDVDGKALY ΞEAQΞKAAΞΞ ADA AΞLADΞ EDAIHDEL Table 2 Alfalfa M-AKNVAIFG LLFSLLLLVP SQIFA ΞES STDAKΞ Oryzae MRTFAPWIL- --SLLGASA- --VAS AADA TAEAPS Yeast MXFSAGAVLS WSSLLLASS- --VFA QQΞA VAPEDS Bovine M-LRRA-LLC --LALTALF- --RAG AGA PDEEDK Rat M-LSRA-LLC --LALAWAA- --RVG ADA LΞEΞDN Mouse MKLRKAWLLV LLLALTQLLA AASAGDAQΞD TSDTΞNATΞΞ ΞΞΞΞDDDDLΞ -FVL- -DW- -AW- --VL- --VL- VKΞΞNGVWVL NDGNFDNFVA DKDTVLLΞFY APWCGHCKQF APΞYΞKIAST TLDNT SLTGD KLATD VLHKG VLKKΞ LKDNDPPIAV AKIDATSASM LASKFDVSGY PTIKILKKGQ AVDYDGSRTQ - NF HDTVKKHDFI WΞFYAPWCG TF ΞTFVKΞHDLV LAΞFFAPWCG - SF NΞYIQSHDLV LAΞFFAPWCG - NF DEALAAHKYL LVΞFYAPWCG NF AΞPAAHNYLL VEFY-APWCG ΞΞIVAKVRΞV SQPDWTPPPΞ VTLSLTKDNF DDWNNADII LVΞFYAPWCG HCKKLAPΞYΞ KAASILSTHΞ PPWLAKVDA NΞΞHNKDLAS ΞNDVKGFPTI HCKALAPKYΞ QAATΞLKΞKN IPL- -VKVDC TΞΞΞA- -LCR DQGVΞGYPTL KCKNMAPEYV KAAETLVΞKN ITL- -AQIDC TΞNQD- -LCM EKNIPGFPSL HCKALAPΞYA KAAGKLKAEG SΞIRLAKVDA TEESD - -LAQ QYGVRGYPTI HCKALAPEYA KAAAKLKAΞG SΞIRLAKVDA TEΞSD- -LAQ QYGVRGYPTI HCKKLAPΞYΞ KAAKΞLSKRS PPIPLAKVDA TΞQTD - -LAK RFDVSGYPTL KIFRNGG- KN IQΞYKGPRΞA ΞGIVΞYLKKQ SGPAS-TΞ K SADDATAFVG KIFRGLDAVK P- -YQGARQT EAIVSYMVKQ SLPAV- SPVT PΞNLΞΞ-IKT KIFKNRDVNN SIDYΞGPRTA ΞAIVQFMIKQ SQPAV-AWA DLPAYL-ANΞ KFFKNGDTAS PKΞYTAGREA DDIVNWLKKR TGPAA-STLS DGAAAΞALVE KFFKNGDTAS PKΞYTAGRΞA DDIVNWLKKR TGPAA-TTLS DTAAAΞSLVD KIFRKG R PFDYNGPRΞK YGIVDYMIΞQ SGPPSKΞILT LKQVQΞFLKD DNKWIVGVF PKFSGΞΞYDN FIALAΞKLRS DYDFAHTLNA KHLPKGDSSV MDKIWIGYI ASDDQTANDI FTTFAΞSQRD NYLFAATSDA SI--AKAΞGV TFVTPVIVQS GKIDADFNAT FYSMΛNKHFN DYDFVSAΞNA DD--DFKLSI SSΞVAVIGFF KDMΞSDSAKQ FFLAAΞV -D DIPFGITSNS DV--FSKYQL SSΞVTVIGFF KDAGSDSAKQ FLLAAEAV-D DIPFGITSNS DV--FSKYQL GDDWI GLF QGDGDPAYLQ YQDAANNLRΞ DYKFHKTFS? ΞIAKFLKVSL 29 table 2 ( continued) SGPWRLFKP FDELFVDS - - - KDFNVEALΞ KFIEESSTPI VTVFNNEPSN KQPSIVLYKD FDEKKATYDG EIEQDALLSW VKTASTPLVG ELGPΞTYSGY YLPSAM- -DE PWYNGKKAD IADADVFEKW LQVEALPYFG ΞIDGSVFAQY DKDGWLFKK FD EGR- - -NNFEGEVTK EKLLDFIKHN QLPLV EFTE DKDGWLFKK FD - - -EGR- - -NNFEGΞITK ΞKLLDFIKHN QLPLVIΞFTΞ GKLVLTHPΞK FQSKYΞPRFH VMDVQGSTEA SAIKDYWKH ALPLVGHRKT HP.FWKFFNS PNAKAMLFIN FTTΞGAESFK TKYHEVAΞQY KQQGV- SFLV ITAGIPLAYI FAΞTKEΞREQ FTΞΞFKFIAE KHKGSINIVT IDAKLYGAHA VΞSGLPLGYL FYNDΞΞELEΞ YKPLFTELAK KNRGLMNFVS IDARKFGRHA QTAPKIFGGE IKTHILLFLP KSVSDYEGKL SNFKKAAESF KGKILFIFID QTAPKIFGGE IKTHILLFLP KSVSDYDGKL SNFKKAAEGF KGKILFIFID SNDAKRYSKR PLVWYYSVD FSFDYRAATQ FWRNKVLEVA KDFPEYTFAI GDVESSQGAF QYFGLKEΞQV PLI - -IIQHN DGKKFFKPN- - -LΞLDQLPT GNLNLDPSKF PAFAIQDPEK NAKY PYDQSKE- - -VKAKDIGK GNLNMK-EQF PLFAIHDMTE DLKYGLPQLS EEAFDELSDK IVLESKAIΞS SDHTDNQRIL ΞFFGLKKΞEC PAVR-LITLE EEMTKYKPES DΞLTAEKITE SDHTDNQRIL EFFGLKKEEC PAVR-LITLE EEMTKYKPES DELTAEKITQ ADEΞDYATEV KDLGL-SESG EDVN-AA LD ESGKKFAMEP EΞFDSDTLRE WLKAYKDGKV EPFVKSEPIP ETNN-EPVKV WGQTLEDW FKSGKNVLIΞ FIQDVLDDKV EPSIKSEAIP ETQΞ-GPVTV WAHSYKDLV LDNEKDVLLE LVKDFLKGDA SPIVKSQEIF ENQD-SSVFQ LVGKNHDEIV NDPKKDVLVL FCHRFLEGKI KPHLMSQELP DDWDKQPVKV LVGKNFEEVA FDΞKKNVFVE FCHHFLEGKI KPHLMSQELP EDWDKQPVKV LVGKNFEEVA FDEKKNVFVE FVTAFKKGKL KPVIKSQPVP KN-NKGPVKV WGKTFDAIV MDPKKDVLIE FYAPWCGHCK QLAPILDEVA SFQS-DADV IAKLDATAN DIPTDTFDVQ FYAPWCGHCK ALAPKYEELA SLYKD- IPEV TIAKIDATAN DV- -PD-SIT YYAPWCGHCK RLAPTYQELA DTYANATSDV LIAKLDHTEN DV- -RGWIE FYAPWCGHCK QLAPIWDKLG ETYKD-HENI VIAKMDSTAN EV- -EAVKVH FYAPWCGHCK QLAPIWDKLG ETYKD-HENI VIAKMDSTAN EV- -EAVKVH FYAPWCGHCK QLEPIYTSLG KKYKG-QKDL VIAKMDATAN DITNDQYKVE GYPTLYFRSA SGK- -LSQYD GGRTKΞDIIE FIE K NKDKTGAAHQ GFPTIKLFAA GAKDSPVEYΞ GSRTVEDLAN FVK E NGKHKVDALE GYPTIVLYPG GKKSESWYQ GSRSLDSLFD FIK Ξ NGHFDVDGKA SFPTLKFFPA SADRTVIDYN GERTLDGFKK FLESGGQDGA GDDDDLEDLΞ SFPTLKFFPA SADRTVIDYN GΞRTLDGFKK FLΞSGRQDGA GDNDDLDLEE GFPTIYFAPS GDKKNPI- - K F E GGNRDLEHLS EVEQPKAAAQ PE AEQPKDEL VDPKKEQESG DATΞTRAASD ΞTETPAATSD DKSEHDEL LYEΞAQΞKAA ΞEAEADAEAΞ ADADAELADE EDAIHDEL EAEEPDLΞED DD QKAVKDEL ALE- PDMEΞD DD QKAVKDEL KF- -ID-EHA TK RSRTKEΞL REFERENCES CITED IN THE SPECIFICATION Sambrook, J. , Fritsch, E. F. & Maniatiε, T. 1989. Molecular Cloning: A Laboratory Manual. 2. edition. Cold Spring Harbor Lab. , Cold Spring Harbor, NY. U.S. Patent No. 4,683,202 Hudson et al, 1989, Practical Immunology, Third edition, Blackwell Scientific Publications. Beaucage and Caruthers, 1981, Tetrahedron Letters 22, 1981, pp. 1859-1869 Matthes et al., 1984, The EMBO J. 3, 1984, pp. 801-805 R.K. Saiki et al. , 1988, Science 239, pp. 487-491 WO 91/17243 EP 238 023 Malardier et al. Gene 78 (1989), pp. 147-156 Woodcock et al., Nucleic Acids Res. (1989) 17:3469-3478 SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: NOVO NORDISK A/S (B) STREET: Novo Alle (C) CITY: Bagsvaerd (E) COUNTRY: DANMARK (F) POSTAL CODE (ZIP) : DK-2880 (G) TELEPHONE: +45 4444 8888 (H) TELEFAX: +45 4449 3256 (ii) TITLE OF INVENTION: PDI Cloning (iii) NUMBER OF SEQUENCES: 3 (iv) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1953 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus oryzae (B) STRAIN: IFO 4177 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 71..444 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 502..880 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 962..1401 (ix) FEATURE: (A) NAME/KEY: exon (B) LOCATION: 1478..1829 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: join(71..444, 502..880, 962..1401, 1478..1829) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: CXXπCCIGTC CCCAIAGACA GTACACACGT CMCCITTGA TATTGTCACA CITGACAAAT 60 TCCCGACACC ATG CGG ACT TTC GCA CCT TGG ATC TTG AGC CIT CTA GGG 109 Met Arg Thr Phe Ala Pro Trp lie Leu Ser Leu Leu Gly 1 5 10 GCT TCT GCT GTA GCT TCT GCT GCC GAT GOG ACT GCC GAA GCT CCC TCC 157 Ala Ser Ala Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser 15 20 25 GAT GTG GTC TOG CTC ACC GGG GAC ACA TTC GAA ACT TTC GTC AAG GAG 205 Asp Val Val Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu 30 35 40 45 CAT GAC CTA GTT TTG GCC GAG TTT TTT GCT CCC TGG TGT GGC CAT TGC 253 His Asp Leu Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys 50 55 60 AAG GCT CTC GCT COG AAA TAC GAG CAG GCC GCC ACT GAG TTA AAG GAA 301 Lys Ala Leu Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu 65 70 75 AAG AAC ATT CCG CTG GTC AAG GTT GAT TGC ACC GAG GAA GAG GCT CIT 349 Lys Asn lie Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu 80 85 90 TGT AGG GAC CAA GGT GIT GAA GGT TAC CCC ACG CTG AAG ATT TTC CGT 397 Cys Arg Asp Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys lie Phe Arg 95 100 105 GGC CIT GAC GCT GTT AAG CCT TAT CAG GGA GCT CGT CAG ACC GAG GC 444 Gly Leu Asp Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala 110 115 120 125 GTAAGTGTCA CCϋUl lϋl AGCCTIGCTC AAATAATAIT GACCGCIAGT ATCAIAG G 502 ATT GTT TCA TAC ATG GTC AAG CAG TCA CTA CCT GCT GTG TCC CCT GTC 550 lie Val Ser Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val 130 135 140 ACC CCA GAA AAC CTC GAA GAG ATC AAG ACT ATG GAC AAG ATT GTC GTT 598 Thr Pro Glu Asn Leu Glu Glu lie Lys Thr Met Asp Lys lie Val Val 145 150 155 ATT GGT TAT ATC GOG TCT GAC GAC CAG ACT GCC AAT GAT ATA TTC ACC 646 lie Gly Tyr lie Ala Ser Asp Asp Gin Thr Ala Asn Asp lie Phe Thr 160 165 170 ACT TTT GCC GAG TCA CAG AGA GAC AAC TAC CTC TTC GCC GCC ACA ACT 694 Thr Phe Ala Glu Ser Gin Arg Asp Asn Tyr Leu Phe Ala Ala Thr Ser 175 180 185 GAT GCA TOG ATC GCT AAG GCA GAA GGT GTT AAG CAA CCT TOG AIT GTT 742 Asp Ala Ser He Ala Lys Ala Glu Gly Val Lys Gin Pro Ser He Val 190 195 200 205 CTC TAT AAA GAC TTC GAT GAA AAG AAA GCT ACT TAT GAT GGA GAG ATT 790 Leu Tyr Lys Asp Phe Asp Glu Lys Lys Ala Thr Tyr Asp Gly Glu He 210 215 220 GAA CAG GAT GCC CTC CTC ACT TGG GTC AAG ACT GCC ACT ACC CCC TTG 838 Glu Gin Asp Ala Leu Leu Ser Trp Val Lys Thr Ala Ser Thr Pro Leu 225 230 235 GTG GGC GAG CTG GGC CCA GAG ACT TAC TCC GGA TAT ATA AOG 880 Val Gly Glu Leu Gly Pro Glu Thr Tyr Ser Gly Tyr He Thr 240 245 250 GTATGTCACA AGACACAATC TCAAIATCGC TTCACAAOGT TTAGTAAAIA AΪCAIGAGTT 940 TCTGACATGG GTI GGTTAA G GCT GGC ATT CCA CTG GCG TAC ATT TTC GCC 991 Ala Gly He Pro Leu Ala Tyr He Phe Ala 255 260 GAA ACC AAA GAA GAG OCT GAG CAG TTC ACC GAG GAG TTC AAG TTC ATC 1039 Glu Thr Lys Glu Glu Arg Glu Gin Phe ϊhr Glu Glu Phe Lys Phe He 265 270 275 GCC GAG AAA CAC AAG GGT TCC ATC AAT ATT GTC ACC ATT GAC GCC AAG 1087 Ala Glu Lys His Lys Gly Ser He Asn He Val Thr He Asp Ala Lys 280 285 290 TTG TAC GGC GCT CAT GCA GGC AAT CTC AAC CTT GAC CCC TCC AAG TTC 1135 Leu Tyr Gly Ala His Ala Gly Asn Leu Asn Leu Asp Pro Ser Lys Phe 295 300 305 CCT GCA TTC GCT ATT CAA GAC CCT GAA AAG AAC GCC AAG TAT CCT TAT 1183 Pro Ala Phe Ala He Gin Asp Pro Glu Lys Asn Ala Lys Tyr Pro Tyr 310 315 320 325 GAC CAG TGG AAG GAA GTC AAG GCC AAG GAT ATC GGT AAA TTC ATC CAA 1231 Asp Gin Ser Lys Glu Val Lys Ala Lys Asp He Gly Lys Phe He Gin 330 335 340 GAC GTT CTT GAT GAT AAA GTA GAG CCA AGC AIT AAG TCT GAG GCT ATT 1279 Asp Val Leu Asp Asp Lys Val Glu Pro Ser He Lys Ser Glu Ala He 345 350 355 CCT GAG ACT CAG GAA GGT CCT GTT ACT GTT GTT GTC GCG CAT TCC TAT 1327 Pro Glu Thr Gin Glu Gly Pro Val Thr Val Val Val Ala His Ser Tyr 360 365 370 AAG GAT CTC GTC CTT GAC AAC GAG AAG GAC GTC CTT CTC GAA TTT TAT 1375 Lys Asp Leu Val Leu Asp Asn Glu Lys Asp Val Leu Leu Glu Phe Tyr 375 380 385 GOG CCA TGG TGC GGA CAC TGC AAG GC GTAAGTITTC CXXXπ?CITTC 1421 Ala Pro Trp Cys Gly His Cys Lys Ala 390 395 TCTACAACGA AΪTAIAICCA CTCTOGCTTG CGAAIACCTA AITAAACC T GAAIAG C 1478 CTT GCC COG AAG TAC GAG GAA CTT GCA AGC CTT TAC AAG GAT ATT CCT 1526 Leu Ala Pro Lys Tyr Glu Glu Leu Ala Ser Leu Tyr Lys Asp He Pro 400 405 410 GAA GTT ACC ATC GCC AAA ATT GAC GCA AOG GCC AAC GAT GTC CCC GAC 1574 Glu Val Thr He Ala Lys He Asp Ala Thr Ala Asn Asp Val Pro Asp 415 420 425 430 TCC ATT ACA GGA TTT CCT ACT ATT AAG CTC TTC GCT GCC GGC GCC AAG 1622 Ser He Thr Gly Phe Pro Thr He Lys Leu Phe Ala Ala Gly Ala Lys 435 440 445 GAC TCC CCA GTT GAA TAT GAA GGC TCT CGC ACG GTG GAG GAC CTC GCC 1670 Asp Ser Pro Val Glu Tyr Glu Gly Ser Arg Thr Val Glu Asp Leu Ala 450 455 460 AAC TTC GTC AAG GAG AAT GGC AAG CAC AAG GTC GAT GCT CTT GAA GTT 1718 Asn Phe Val Lys Glu Asn Gly Lys His Lys Val Asp Ala Leu Glu Val 465 470 475 GAT COG AAG AAA GAA CAG GAG ACT GGC GAT GCC ACC GAG ACT CGG GCC 1766 Asp Pro Lys Lys Glu Gin Glu Ser Gly Asp Ala Thr Glu Thr Arg Ala 480 485 490 GCC TCT GAC GAG ACC GAA ACT CCT GCT GCT ACT AGC GAT GAC AAG TCT 1814 Ala Ser Asp Glu Thr Glu Thr Pro Ala Ala Thr Ser Asp Asp Lys Ser 495 500 505 510 GAG CAT GAT GAA TTG TAAATTTCAT TTGGCCTGAT AGTTTGAICC A_EOTIATGT 1869 Glu His Asp Glu Leu 515 GAATTCTTCT ATTCTACCAG OVGTTTGAGC AATCGCAGCT ACTTCOGGCT TAGGAAACTG 1929 TTGTTCTATC CTAGTGGGAA GCTT 1953 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1548 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iii) ANTI-SENSE: NO (vi) ORIGINAL SOURCE: (A) ORGANISM: Aspergillus oryzae (B) STRAIN: IFO 4177 (ix) FEATURE: (A) NAME/KEY: CDS (B) LOCATION: 1..1548 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: AIG CGG ACT TTC GCA CCT TGG ATC TTG AGC CTT CTA GGG GCT TCT GCT 48 Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala - 1 5 10 15 GTA GCT TCT GCT GCC GAT GCG ACT GCC GAA GCT CCC TCC GAT GTG GTC 96 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 TOG CTC ACC GGG GAC ACA TTC GAA ACT TTC GTC AAG GAG CAT GAC CTA 144 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 GTT TTG GCC GAG TTT TTT GCT CCC TGG TGT GGC CAT TGC AAG GCT CTC 192 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 GCT COG AAA TAC GAG CAG GCC GCC ACT GAG TTA AAG GAA AAG AAC ATT 240 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 COG CTG GTC AAG GTT GAT TGC ACC GAG GAA GAG GCT CTT TGT AGG GAC 288 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 CAA GGT GTT GAA GGT TAC CCC ACG CTG AAG ATT TTC OGT GGC CTT GAC 336 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 GCT GTT AAG CCT TAT CAG GGA GCT CGT CAG ACC GAG GCG ATT CTT TCA 384 Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 TAC ATG GTC AAG CAG TCA CTA CCT GCT GTG TCC CCT GTC ACC CCA GAA 432 Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val Thr Pro Glu 130 135 140 AAC CTC GAA GAG ATC AAG ACT ATG GAC AAG ATT GTC GTT AIT GCT TAT 480 Asn Leu Glu Glu He Lys Thr Met Asp Lys He Val Val He Gly Tyr 145 150 155 160 ATC GCG TCT GAC GAC CAG ACT GCC AAT GAT ATA TTC ACC ACT TTT GCC 528 He Ala Ser Asp Asp Gin Thr Ala Asn Asp He Phe Thr Thr Phe Ala 165 170 175 GAG TCA CAG AGA GAC AAC TAC CTC TTC GCC GCC ACA ACT GAT GCA TOG 576 Glu Ser Gin Arg Asp Asn Tyr Leu Phe Ala Ala Thr Ser Asp Ala Ser 180 185 190 ATC GCT AAG GCA GAA GCT GTT AAG CAA CCT TCG ATT GTT CTC TAT AAA 624 He Ala Lys Ala Glu Gly Val Lys Gin Pro Ser He Val Leu Tyr Lys 195 200 205 GAC TTC GAT GAA AAG AAA GCT ACT TAT GAT GGA GAG ATT GAA CAG GAT 672 Asp Phe Asp Glu Lys Lys Ala Thr Tyr Asp Gly Glu He Glu Gin Asp 210 215 220 GCC CTC CTC ACT TGG GTC AAG ACT GCC ACT ACC CCC TTG GTG GGC GAG 720 Ala Leu Leu Ser Trp Val Lys Thr Ala Ser Thr Pro Leu Val Gly Glu 225 230 235 240 CTG GGC CCA GAG ACT TAC TCC GGA TAT ATA ACG GCT GGC AIT CCA CTG 768 Leu Gly Pro Glu Thr Tyr Ser Gly Tyr He Thr Ala Gly He Pro Leu 245 250 255 GCG TAC ATT TTC GCC GAA ACC AAA GAA GAG CGT GAG CAG TTC ACC GAG 816 Ala Tyr He Phe Ala Glu Thr Lys Glu Glu Arg Glu Gin Phe Thr Glu 260 265 270 GAG TTC AAG TTC ATC GCC GAG AAA CAC AAG GCT TCC ATC AAT ATT GTC 864 Glu Phe Lys Phe He Ala Glu Lys His Lys Gly Ser He Asn He Val 275 280 285 ACC ATT GAC GCC AAG TTG TAC GGC GCT CAT GCA GGC AAT CTC AAC CTT 912 Thr He Asp Ala Lys Leu Tyr Gly Ala His Ala Gly Asn Leu Asn Leu 290 295 300 GAC CCC TCC AAG TTC CCT GCA TTC GCT ATT CAA GAC CCT GAA AAG AAC 960 Asp Pro Ser Lys Phe Pro Ala Phe Ala He Gin Asp Pro Glu Lys Asn 305 310 315 320 GCC AAG TAT CCT TAT GAC CAG TCG AAG GAA GTC AAG GCC AAG GAT ATC 1008 Ala Lys Tyr Pro Tyr Asp Gin Ser Lys Glu Val Lys Ala Lys Asp He 325 -330 335 GGT AAA TTC ATC CAA GAC GTT CTT GAT GAT AAA GTA GAG CCA AGC ATT 1056 Gly Lys Phe He Gin Asp Val Leu Asp Asp Lys Val Glu Pro Ser He 340 345 350 AAG TCT GAG GCT AIT CCT GAG ACT CAG GAA GGT CCT GTT ACT GTT GTT 1104 Lys Ser Glu Ala He Pro Glu Thr Gin Glu Gly Pro Val Thr Val Val 355 360 365 GTC GCG CAT TCC TAT AAG GAT CTC GTC CTT GAC AAC GAG AAG GAC GTC 1152 Val Ala His Ser Tyr Lys Asp Leu Val Leu Asp Asn Glu Lys Asp Val 370 375 380 CTT CTC GAA TTT TAT GCG CCA TGG TGC GGA CAC TGC AAG GCC CTT GCC 1200 Leu Leu Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala 385 390 395 400 COG AAG TAC GAG GAA CTT GCA AGC CTT TAC AAG GAT ATT CCT GAA GTT 1248 Pro Lys Tyr Glu Glu Leu Ala Ser Leu Tyr Lys Asp He Pro Glu Val 405 410 415 ACC ATC GCC AAA ATT GAC GCA ACG GCC AAC GAT GTC CCC GAC TCC ATT 1296 Thr He Ala Lys He Asp Ala Thr Ala Asn Asp Val Pro Asp Ser He 420 425 430 ACA GGA TTT CCT ACT ATT AAG CTC TTC GCT GCC GGC GCC AAG GAC TCC 1344 Thr Gly Phe Pro Thr He Lys Leu Phe Ala Ala Gly Ala Lys Asp Ser 435 440 445 CCA GTT GAA TAT GAA GGC TCT CGC ACG GTG GAG GAC CTC GCC AAC TTC 1392 Pro Val Glu Tyr Glu Gly Ser Arg Thr Val Glu Asp Leu Ala Asn Phe 450 455 460 GTC AAG GAG AAT GGC AAG CAC AAG GTC GAT GCT CTT GAA GTT GAT COG 1440 Val Lys Glu Asn Gly Lys His Lys Val Asp Ala Leu Glu Val Asp Pro 465 470 475 480 AAG AAA GAA CAG GAG ACT GGC GAT GCC ACC GAG ACT CGG GCC GCC TCT 1488 Lys Lys Glu Gin Glu Ser Gly Asp Ala Thr Glu Thr Arg Ala Ala Ser 485 490 495 GAC GAG ACC GAA ACT CCT GCT GCT ACT AGC GAT GAC AAG TCT GAG CAT 1536 Asp Glu Thr Glu Thr Pro Ala Ala Thr Ser Asp Asp Lys Ser Glu His 500 505 510 GAT GAA TTG TA 1548 Asp Glu Leu 515 (2 ) INFORMATION FOR SEQ ID NO : 3 : ( i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 515 amino acids (B) TYPE : amino acid (D) TOPOLOGY : linear (ii) MOLECULE TYPE : protein ( i) SEQUENCE DESCRIPTION: SEQ ID NO : 3 : Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val Thr Pro Glu 130 135 140 Asn Leu Glu Glu He Lys Thr Met Asp Lys He Val Val He Gly Tyr 145 150 155 160 He Ala Ser Asp Asp Gin Thr Ala Asn Asp He Phe Thr Thr Phe Ala 165 170 175 Glu Ser Gin Arg Asp Asn Tyr Leu Phe Ala Ala Thr Ser Asp Ala Ser 180 185 190 He Ala Lys Ala Glu Gly Val Lys Gin Pro Ser He Val Leu Tyr Lys 195 200 205 Asp Phe Asp Glu Lys Lys Ala Thr Tyr Asp Gly Glu He Glu Gin Asp 210 215 220 Ala Leu Leu Ser Trp Val Lys Thr Ala Ser Thr Pro Leu Val Gly Glu 225 230 235 240 Leu Gly Pro Glu Thr Tyr Ser Gly Tyr He Thr Ala Gly He Pro Leu 245 250 255 Ala Tyr He Phe Ala Glu Thr Lys Glu Glu Arg Glu Gin Phe Thr Glu 260 265 270 Glu Phe Lys Phe He Ala Glu Lys His Lys Gly Ser He Asn He Val 275 280 285 Thr He Asp Ala Lys Leu Tyr Gly Ala His Ala Gly Asn Leu Asn Leu 290 295 300 Asp Pro Ser Lys Phe Pro Ala Phe Ala He Gin Asp Pro Glu Lys Asn 305 310 315 320 Ala Lys Tyr Pro Tyr Asp Gin Ser Lys Glu Val Lys Ala Lys Asp He 325 330 335 Gly Lys Phe He Gin Asp Val Leu Asp Asp Lys Val Glu Pro Ser He 340 345 350 Lys Ser Glu Ala He Pro Glu Thr Gin Glu Gly Pro Val Thr Val Val 355 360 365 Val Ala His Ser Tyr Lys Asp Leu Val Leu Asp Asn Glu Lys Asp Val 370 375 380 Leu Leu Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala 385 390 395 400 Pro Lys Tyr Glu Glu Leu Ala Ser Leu Tyr Lys Asp He Pro Glu Val 405 410 415 Thr He Ala Lys He Asp Ala Thr Ala Asn Asp Val Pro Asp Ser He 420 425 430 Thr Gly Phe Pro Thr He Lys Leu Phe Ala Ala Gly Ala Lys Asp Ser 435 440 445 Pro Val Glu T r Glu Gly Ser Arg Thr Val Glu Asp Leu Ala Asn Phe 450 455 460 Val Lys Glu Asn Gly Lys His Lys Val Asp Ala Leu Glu Val Asp Pro 465 470 475 480 Lys Lys Glu Gin Glu Ser Gly Asp Ala Thr Glu Thr Arg Ala Ala Ser 485 490 495 Asp Glu Thr Glu Thr Pro Ala Ala Thr Ser Asp Asp Lys Ser Glu His 500 505 510 Asp Glu Leu 515 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 511 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Tϊp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val Thr Pro Glu 130 135 140 Asn Leu Glu Glu He Lys Thr Met Asp Lys He Val Val He Gly Tyr 145 150 155 160 He Ala Ser Asp Asp Gin Thr Ala Asn Asp He Phe Thr Thr Phe Ala 165 170 175 Glu Ser Gin Arg Asp Asn Tyr Leu Phe Ala Ala Thr Ser Asp Ala Ser 180 185 190 He Ala Lys Ala Glu Gly Val Lys Gin Pro Ser He Val Leu Tyr Lys 195 200 205 Asp Phe Asp Glu Lys Lys Ala Thr Tyr Asp Gly Glu He Glu Gin Asp 210 215 220 Ala Leu Leu Ser Trp Val Lys Thr Ala Ser Thr Pro Leu Val Gly Glu 225 230 235 240 Leu Gly Pro Glu Thr Tyr Ser Gly Tyr He Thr Ala Gly He Pro Leu 245 250 255 Ala Tyr He Phe Ala Glu Thr Lys Glu Glu Arg Glu Gin Phe Thr Glu 260 265 270 Glu Phe Lys Phe He Ala Glu Lys His Lys Gly Ser He Asn He Val 275 280 285 Thr He Asp Ala Lys Leu Tyr Gly Ala His Ala Gly Asn Leu Asn Leu 290 295 300 Asp Pro Ser Lys Phe Pro Ala Phe Ala He Gin Asp Pro Glu Lys Asn 305 310 315 320 Ala Lys Tyr Pro Tyr Asp Gin Ser Lys Glu Val Lys Ala Lys Asp He 325 330 335 Gly Lys Phe He Gin Asp Val Leu Asp Asp Lys Val Glu Pro Ser He 340 345 350 Lys Ser Glu Ala He Pro Glu Thr Gin Glu Gly Pro Val Thr Val Val 355 360 365 Val Ala His Ser Tyr Lys Asp Leu Val Leu Asp Asn Glu Lys Asp Val 370 375 380 Leu Leu Glu Phe Tyr Ala Pro Trp Cys Gly His Cys Lys Ala Leu Ala 385 390 395 400 Pro Lys Tyr Glu Glu Leu Ala Ser Leu Tyr Lys Asp He Pro Glu Val 405 410 415 Thr He Ala Lys He Asp Ala Thr Ala Asn Asp Val Pro Asp Ser He 420 425 430 Thr Gly Phe Pro Thr He Lys Leu Phe Ala Ala Gly Ala Lys Asp Ser 435 440 445 Pro Val Glu Tyr Glu Gly Ser Arg Thr Val Glu Asp Leu Ala Asn Phe 450 455 460 Val Lys Glu Asn Gly Lys His Lys Val Asp Ala Leu Glu Val Asp Pro 465 470 475 480 Lys Lys Glu Gin Glu Ser Gly Asp Ala Thr Glu Thr Arg Ala Ala Ser 485 490 495 Asp Glu Thr Glu Thr Pro Ala Ala Thr Ser Asp Asp Lys Ser Ala 500 505 510 ( 2 ) INFORMATION FOR SEQ ID NO : 5 : ( i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 281 amino acids (B) TYPE: amino acid (D) TOPOLOGY : linear ( ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 5 : Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val Thr Pro Glu 130 135 140 Asn Leu Glu Glu He Lys Thr Met Asp Lys He Val Val He Gly Tyr 145 150 155 160 He Ala Ser Asp Asp Gin Thr Ala Asn Asp He Phe Thr Thr Phe Ala 165 170 175 Glu Ser Gin Arg Asp Asn Tyr Leu Phe Ala Ala Thr Ser Asp Ala Ser 180 185 190 He Ala Lys Ala Glu Gly Val Lys Gin Pro Ser He Val Leu Tyr Lys 195 200 205 Asp Phe Asp Glu Lys Lys Ala Thr Tyr Asp Gly Glu He Glu Gin Asp 210 215 220 Ala Leu Leu Ser Trp Val Lys Thr Ala Ser Thr Pro Leu Val Gly Glu 225 230 235 240 Leu Gly Pro Glu Thr Tyr Ser Gly Tyr He Thr Ala Gly He Pro Leu 245 250 255 Ala Tyr He Phe Ala Glu Thr Lys Glu Glu Arg Glu Gin Phe Thr Glu 260 265 270 Glu Phe Lys Phe He Ala Glu Lys His 275 280 ( 2 ) INFORMATION FOR SEQ ID NO : 6 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 174 amino acids (B) TYPE : amino acid (D) TOPOLOGY : linear ( ii ) MOLECULE TYPE : protein (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 6 : Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 HO Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val Thr Pro Glu 130 135 140 Asn Leu Glu Glu He Lys Thr Met Asp Lys He Val Val He Gly Tyr 145 150 155 160 He Ala Ser Asp Asp Gin Thr Ala Asn Asp He Phe Thr Thr 165 170 ( 2 ) INFORMATION FOR SEQ ID NO : 7 : ( i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 163 amino acids (B) TYPE : amino acid (D) TOPOLOGY : linear ( ii) MOLECULE TYPE : protein (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 7 : Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val Thr Pro Glu 130 135 140 Asn Leu Glu Glu He Lys Thr Met Asp Lys He Val Val He Gly Tyr 145 150 155 160 He Ala Ser (2) INFORMATION FOR SEQ ID NO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 143 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (Xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val Thr Pro 130 135 140 (2 ) INFORMATION FOR SEQ ID NO : 9 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH : 141 amino acids (B) TYPE : amino acid ( D) TOPOLOGY : linear ( ii ) MOLECULE TYPE : protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 Tyr Met Val Lys Gin Ser Leu Pro Ala Val Ser Pro Val 130 135 140 ( 2 ) INFORMATION FOR SEQ ID NO : 10 : ( i) SEQUENCE CHARACTERISTICS : (A) LENGTH : 131 amino acids (B) TYPE: amino acid (D) TOPOLOGY : linear ( ii ) MOLECULE TYPE : protein (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 10 : Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 Ala Val Lys Pro Tyr Gin Gly Ala Arg Gin Thr Glu Ala He Val Ser 115 120 125 Tyr Met Val 130 (2) INFORMATION FOR SEQ ID NO: 11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 129 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: Met Arg Thr Phe Ala Pro Trp He Leu Ser Leu Leu Gly Ala Ser Ala 1 5 10 15 Val Ala Ser Ala Ala Asp Ala Thr Ala Glu Ala Pro Ser Asp Val Val 20 25 30 Ser Leu Thr Gly Asp Thr Phe Glu Thr Phe Val Lys Glu His Asp Leu 35 40 45 Val Leu Ala Glu Phe Phe Ala Pro Trp Cys Gly His Cys Lys Ala Leu 50 55 60 Ala Pro Lys Tyr Glu Gin Ala Ala Thr Glu Leu Lys Glu Lys Asn He 65 70 75 80 Pro Leu Val Lys Val Asp Cys Thr Glu Glu Glu Ala Leu Cys Arg Asp 85 90 95 Gin Gly Val Glu Gly Tyr Pro Thr Leu Lys He Phe Arg Gly Leu Asp 100 105 110 Ala Val Lys Leu He Arg Glu Leu Leu Gin Glu Leu Val Asn Lys His 115 120 125 Leu (2) INFORMATION FOR SEQ ID NO: 12: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 200 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION : SEQ ID NO : 12 : Ala Glu Ala Pro Ser Asp Val Val Ser Leu Thr Gly Asp Thr Phe Glu 1 5 10 15 Thr Phe Val Lys Glu His Asp Leu Val Leu Ala Glu Phe Phe Ala Pro 20 25 30 Trp Cys Gly His Cys Lys Ala Leu Ala Pro Lys Tyr Glu Gin Ala Ala 35 40 45 Thr Glu Leu Lys Glu Lys Asn He Pro Leu Val Lys Val Asp Cys Thr Glu 50 55 60 Glu Glu Ala Leu Cys Arg Asp Gin Gly Val Glu Gly Tyr Pro Thr 65 70 75 80 Leu Lys He Phe Arg Gly Leu Asp Ala Val Lys Pro Tyr Gin Gly Ala 85 90 95 Arg Gin Thr Glu Ala He Val Ser Tyr Met Val Lys Gin Ser Leu Pro 100 105 110 Ala Val Ser Pro Val Thr Pro Glu Asn Leu Glu Glu He Lys Thr Met H5 120 125 Asp Lys He Val Val He Gly Tyr He Ala Ser Asp Asp Gin Thr Ala 130 135 140 Asn Asp He Phe Thr Thr Phe Ala Glu Ser Gin Arg Asp Asn Tyr Leu 145 150 155 160 Phe Ala Ala Thr Ser Asp Ala Ser He Ala Lys Ala Glu Gly Val Lys 165 170 175 Gin Pro Ser He Val Leu Tyr Lys Asp Phe Asp Glu Lys Lys Ala Thr 180 185 190 Tyr Asp Gly Glu He Glu Gin Asp 195 200 ( 2 ) INFORMATION FOR SEQ ID NO : 13 : ( i ) SEQUENCE CHARACTERISTICS : (A) LENGTH: 25 base pairs (B) TYPE : nucleic acid (C) STRANDEDNESS : single (D) TOPOLOGY : linear ( ii ) MOLECULE TYPE : primer 4762 ( iii) HYPOTHETICAL : YES ( iii) ANTI-SENSE : NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13: TGGAATTCTG GTGYGGNCAY TGYAA 25 (2) INFORMATION FOR SEQ ID NO: 14: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 4763 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TGGGATCCRC ACCANGGNGC RTA 23 (2) INFORMATION FOR SEQ ID NO: 15: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 5205 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TTCGGATCCA CCATGCGGAC TTTCGCACC 29 (2) INFORMATION FOR SEQ ID NO: 16: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 55 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 5215 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: CCAAGCTTTA GAGATGCTTG TTGACAAGCT CCTGGAGGAG CTCCCTGATA AGCTT 55 (2) INFORMATION FOR SEQ ID NO: 17: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 5397 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: CCAAGCTTTA GACCATGTAT GACACAATCG CCTCGGTCTG ACGAG 55 (2) INFORMATION FOR SEQ ID NO: 18: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 5895 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: CCAAGCTTTA GACAGGGGAC ACAGCAGGTA G 31 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 5399 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 19: CCAAGCTTTA TGGGGTGACA GGGGACA 27 (2) INFORMATION FOR SEQ ID NO: 20: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 5894 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: CCAAGCTTTA AGACGCGATA TAACCAATAA C 31 (2) INFORMATION FOR SEQ ID NO: 21: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 5893 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: CCAAGCTTTA AGTGGTGAAT ATATCATTGG C 31 (2) INFORMATION FOR SEQ ID NO: 22: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 6314 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 22: CCAAGCTTAG TGTTTCTCGG CGATGAACTT (2) INFORMATION FOR SEQ ID NO: 23: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: primer 5204 (iii) HYPOTHETICAL: YES (iii) ANTI-SENSE: NO (xi) ' SEQUENCE DESCRIPTION: SEQ ID NO: 23: CCAAGCTTTA CGCAGACTTG TCATCGCTAG T 31

Claims: PATENT CLAIMS 1. An enzyme exhibiting protein disulfide isomerase activity, which enzyme is producible by a filamentous fungus. 2. The enzyme of claim 1, wherein said filamentous fungus belongs to the genus Aεpergilluε . 3. The enzyme of claim 2, wherein said Aεpergillus is A. oryzae or A. niger. 4. An enzyme exhibiting protein disulfide isomerase activity, which enzyme is immunologically reactive with an antibody raised against a purified protein disulfide isomerase derived from A. oryzae , IFO 4177 or A. niger 524 (ATCC 16882) . 5. The enzyme of claim 4, which enzyme is immunologically reactive with an antibody raised against a purified protein disulfide isomerase derived from A. oryzae , IFO 4177. 6. The enzyme of claim 5 having an amino acid sequence as shown in SEQ ID No. 3. 7. An active protein disulfide isomerase enzyme being a truncated form of an enzyme according to any of the claims 1 to 6 comprising an amino acid corresponding to residues 1-131(SEQ ID No. 10), 1-141(SEQ ID No. 9), 1-143(SEQ ID No. 8), 1-163(SEQ ID No. 7), 1-174(SEQ ID No. 6), 1-281(SEQ ID No. 5), or 25- 224(SEQ ID No. 12) of the amino acid sequence of the original enzyme(SEQ ID No. 3). 8. An active protein disulfide isomerase enzyme being a truncated form of an enzyme according to any of the claims l to 6 comprising an amino acid corresponding to residues 1-115 and extended with a sequence Leu-Ile-Arg-Glu-Leu-Leu-Gln-Glu-Leu- Val-Asn-Lys-His-Leu (SEQ ID No. 11) , or corresponding to resi¬ dues 1-510 and extended with an Ala residue. 9. An active protein disulfide isomerase enzyme being a truncated form of an enzyme according to any of the claims 1 to 6 comprising an amino acid corresponding to residues 1-514 and extended with the residue Ala(SEQ ID No. 10). 10. An active protein disulfide isomerase enzyme characterized by exhibiting a homology of at least 60%, preferably 70%, more preferable 80% in the amino acid sequence when compared to any enzyme according to claims 1 to 9. 11. A DNA construct encoding a protein disulfide isomerase producible by a filamentous fungi. 12. The DNA construct of claim 11, wherein said filamentous fungus belongs to the genus Aεpergilluε . 13. The DNA construct of claim 12, wherein said Aεpergilluε is Aspergillus niger or Aεpergilluε oryzae . 14. A DNA construct comprising a DNA sequence encoding the recombinant protein disulfide isomerase enzyme as claimed in any of claims 1 to 10. 15. The DNA construct according to any of the claims 11 to 14, in which the DNA sequence is derivable from a filamentous fungus. 16. The DNA construct according to claim 15, in which the DNA sequence is derivable from a strain of a Aspergillus species, in particular a strain of A. oryzae or A. niger. 17. The DNA construct according to any of the claims 11 to 16, wherein the DNA hybridises to the same probe as the DNA coding for the protein disulfide isomerase as shown in the appended SEQ ID No. 1 or 2, and coding for a polypeptide exhibiting protein disulfide isomerase activity. 18. The DNA construct according to any of the claims 11 to 17, wherein the DNA sequence is as shown in the appended SEQ ID No. 1 or SEQ ID No. 2. 19. The DNA construct according to any of the claims 11 to 18, wherein the DNA sequence is degenerate as a result of the genetic code to the DNA sequences indicated in claims 12 or 13. 20. A recombinant expression vector comprising a DNA construct according to any of claims 11-19. 21. The vector of claim 20 wherein said DNA construct is operably linked to a promoter sequence and optionally to a sequence encoding a secretion signal. 22. A cell comprising a DNA construct according to any of claims 11-19 or a vector according to any of claims 20 or 21. 23. A cell according to claim 22, which is a microbial cell. 24. The cell according to claim 23, which is a bacterial cell or a fungal cell. 25. The cell according to claim 24, in which the bacterial cell is a cell of a gram-positive bacterium, e.g. of the genus Bacillus or Streptomyces or a cell of a gram-negative bacte¬ rium, e.g. of the genus Escherichia , and the fungal cell is a yeast cell, e.g. of the genus Saccharomyceε , or a cell of a filamentous fungus, e.g. of the genus Aspergillus or Fusarium . 26. A method of producing a protein disulfide isomerase enzyme as defined in any of the claims 1 to 10, wherein a cell from a strain of an Aspergillus species, in particular a strain of A. oryzae or A. niger is cultured under conditions conducive to the production of the protein disulfide isomerase, and the protein disulfide isomerase is subsequently recovered from the culture. 27. A method of producing a recombinant protein disulfide isomerase enzyme as defined in any of the claims 1 to 7, wherein a cell according to any of claims 18 to 22 is cultured under conditions conducive to the production of the protein disulfide isomerase, and the protein disulfide isomerase is subsequently recovered from the culture. 28. The method according to claim 27, in which the recombinant protein disulfide isomerase enzyme is expressed in the form of a proenzyme and the cell is cultured in the presence of a proteolytic enzyme capable of converting the proenzyme of the protein disulfide isomerase into a mature enzyme. 29. A composition comprising a recombinant protein disulfide isomerase enzyme as defined in any of the claims 1 to 10. 30. The composition according to claim 29, in the form of a non-dusting granulate, stabilised liquid or protected enzyme. 31. The composition according to claim 29 or 27 which contains 0.01-200 mg of enzyme protein/g. 32. The composition according to claim 31 which contains 0.01- 20 mg of enzyme protein/g. 33. The composition according to claim 31 which contains 0.01-2 mg of enzyme protein/g. 34. The composition according to claim 31 which contains 0.02- 0.2 mg of enzyme protein/g. 35. The composition according to claim 31 which contains 0.01- 0.2 mg of enzyme protein/g. 36. The composition according to any of the claims 29 to 35 which additionally comprises another enzyme such as a protease, an amylase, a lipase, a peroxidase and/or a cellulase. 37. The composition according to any of the claims 29 to 36 which is a pharmaceutical composition. 38. A process for treating scleroproteins which comprises applying any of the compositions of any of claims 29 to 36 to the scleroprotein. 39. The process of claim 38, wherein said scleroprotein is human hair or skin; or animal hair or skin. 40. The process of claim 39, wherein said process involves waving, straightening, removing, degrading or softening of said hair; or softening and/or restoration of said skin. 41. A process for dehairing and/or softening of hides involving the application of any of the compositions of claims 29 to 36 to said hides. 42. A process for the cleaning of fabrics involving the application of any of the compositions of claims 29 to 36 to said fabrics. 43. The process of claim 42 also involving treatment with a detergent. 44. A process for thickening and/or gelation of food and/or fodder products involving application of any of the composi¬ tions of claims 29 to 36 to the food or fodder. 45. A process for strengthening of gluten in bakery or pastry products involving application of any of the compositions of claims 29 to 36 in the process of producing said bakery or pastry products. 46. A process for the treatment of eye conditions, such as cataracts, involving application of the composition of claim 37 to the eye.

Inventors: HJORT CARSTEN M, HJORT, CARSTEN, M.
Applicants: HJORT CARSTEN M, NOVONORDISK AS, HJORT, CARSTEN, M., NOVO NORDISK A/S
Application year: 1994
Main classification: C12N9
IPC codes: A21D8, A23B5, A23K1, A23L1, A61K8, A61K38, A61Q5, C12N1, C12N9, C12N15, C12R1, D06M16